Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the~120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.

The metabolism of one-carbon (C1) units is essential to plants, and plant C1 metabolism has novel features not found in other organisms-plus some enigmas. Despite its centrality, uniqueness, and mystery, plant C1 biochemistry has historically been quite poorly explored, in part because its enzymes and intermediates tend to be labile and low in abundance. Fortunately, the integration of molecular and genetic approaches with biochemical ones is now driving rapid advances in knowledge of plant C1 enzymes and genes. An overview of these advances is presented. There has also been progress in measuring C1 metabolite fluxes and pool sizes, although this remains challenging and there are relatively few data. In the future, combining reverse genetics with flux and pool size determinations should lead to quantitative understanding of how plant C1 pathways function. This is a prerequisite for their rational engineering.

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All flowering plants produce S-methylmethionine (SMM) from Met and have a separate mechanism to convert SMM back to Met. The functions of SMM and the reasons for its interconversion with Met are not known. In this study, by using the aphid stylet collection method together with mass spectral and radiolabeling analyses, we established that l-SMM is a major constituent of the phloem sap moving to wheat ears. The SMM level in the phloem ( approximately 2% of free amino acids) was 1.5-fold that of glutathione, indicating that SMM could contribute approximately half the sulfur needed for grain protein synthesis. Similarly, l-SMM was a prominently labeled product in phloem exudates obtained by EDTA treatment of detached leaves from plants of the Poaceae, Fabaceae, Asteraceae, Brassicaceae, and Cucurbitaceae that were given l-(35)S-Met. cDNA clones for the enzyme that catalyzes SMM synthesis (S-adenosylMet:Met S-methyltransferase; EC 2.1.1.12) were isolated from Wollastonia biflora, maize, and Arabidopsis. The deduced amino acid sequences revealed the expected methyltransferase domain ( approximately 300 residues at the N terminus), plus an 800-residue C-terminal region sharing significant similarity with aminotransferases and other pyridoxal 5'-phosphate-dependent enzymes. These results indicate that SMM has a previously unrecognized but often major role in sulfur transport in flowering plants and that evolution of SMM synthesis in this group involved a gene fusion event. The resulting bipartite enzyme is unlike any other known methyltransferase.

Regulation of transcriptional processes is a critical mechanism that enables efficient coordination of the synthesis of required proteins in response to environmental and cellular changes. Transcription factors require accurate activity regulation because they play a critical role as key mediators assuring specific expression of target genes. In this work, we show that CULLIN3-based E3 ligases have the potential to interact with a broad range of ETHYLENE RESPONSE FACTOR (ERF)/APETALA2 (AP2) transcription factors, mediated by MATH-BTB/POZ (for Meprin and TRAF [tumor necrosis factor receptor associated factor] homolog)-Broad complex, Tramtrack, Bric-a-brac/Pox virus and Zinc finger) proteins. The assembly with an E3 ligase causes degradation of their substrates via the 26S proteasome, as demonstrated for the WRINKLED1 ERF/AP2 protein. Furthermore, loss of MATH-BTB/POZ proteins widely affects plant development and causes altered fatty acid contents in mutant seeds. Overall, this work demonstrates a link between fatty acid metabolism and E3 ligase activities in plants and establishes CUL3-based E3 ligases as key regulators in transcriptional processes that involve ERF/AP2 family members.

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All flowering plants produce S -methylmethionine (SMM) from Met and have a separate mechanism to convert SMM back to Met. The functions of SMM and the reasons for its interconversion with Met are not known. In this study, by using the aphid stylet collection method together with mass spectral and radiolabeling analyses, we established that L -SMM is a major constituent of the phloem sap moving to wheat ears. The SMM level in the phloem ( ‫ف‬ 2% of free amino acids) was 1.5-fold that of glutathione, indicating that SMM could contribute approximately half the sulfur needed for grain protein synthesis. Similarly, L -SMM was a prominently labeled product in phloem exudates obtained by EDTA treatment of detached leaves from plants of the Poaceae, Fabaceae, Asteraceae, Brassicaceae, and Cucurbitaceae that were given L -35 S-Met. cDNA clones for the enzyme that catalyzes SMM synthesis ( S -adenosylMet:Met S -methyltransferase; EC 2.1.1.12) were isolated from Wollastonia biflora , maize, and Arabidopsis. The deduced amino acid sequences revealed the expected methyltransferase domain ( ‫ف‬ 300 residues at the N terminus), plus an 800-residue C-terminal region sharing significant similarity with aminotransferases and other pyridoxal 5 -phosphate-dependent enzymes. These results indicate that SMM has a previously unrecognized but often major role in sulfur transport in flowering plants and that evolution of SMM synthesis in this group involved a gene fusion event. The resulting bipartite enzyme is unlike any other known methyltransferase. INTRODUCTIONPlant Met metabolism differs from that in other organisms by involving S -methylmethionine (SMM). SMM is a ubiquitous constituent of the free amino acid pool in flowering plants, occurring in leaves, roots, and other organs at levels that typically range from 0.5 to 3 mol g Ϫ 1 dry weight, a concentration that is often higher than those of Met or S -adenosylmethionine (AdoMet) (Giovanelli et al., 1980;Mudd and Datko, 1990;Bezzubov and Gessler, 1992). SMM also has been detected as a metabolite of radiolabeled L -Met in all flowering plants tested ( Ͼ 50 species from Ͼ 20 families; Paquet et al., 1995). As shown in Figure 1, SMM is formed from L -Met via the action of AdoMet:Met S -methyltransferase (MMT; EC 2.1.1.12) and can be reconverted to Met by donating a methyl group to L -homocysteine (Hcy) in a reaction catalyzed by Hcy S -methyltransferase (HMT; EC 2.1.1.10; Giovanelli et al., 1980;Mudd and Datko, 1990). The tandem action of MMT and HMT, together with S -adenosyl-L -Hcy hydrolase, constitutes the SMM cycle, which is apparently futile (Mudd and Datko, 1990).As expected from the universality of SMM, MMT activity has been found in many flowering plants (Giovanelli et al., 1980;Mudd and Datko, 1990). It has been purified from leaves of Wollastonia biflora (James et al., 1995a) and from germinating barley (Pimenta et al., 1998), and it is known to have subunits of ‫ف‬ 115 kD. Because this is approximately three times larger than any other small-molecule methyltransferase (F...

Plants synthesize p-aminobenzoate (pABA) in chloroplasts and use it for folate synthesis in mitochondria. It has generally been supposed that pABA exists as the free acid in plant cells and that it moves between organelles in this form. Here we show that fruits and leaves of tomato and leaves of a diverse range of other plants have a high capacity to convert exogenously supplied pABA to its ␤-D-glucopyranosyl ester (pABA-Glc), whereas yeast and Escherichia coli do not. High performance liquid chromatography analysis indicated that much of the endogenous pABA in fruit and leaf tissues is esterified and that the total pool of pABA (free plus esterified) varies greatly between tissues (from 0.2 to 11 nmol g ؊1 of fresh weight). UDP-glucose:pABA glucosyltransferase activity was readily detected in fruit and leaf extracts, and the reaction was found to be freely reversible. p-Aminobenzoic acid ␤-D-glucopyranosyl ester esterase activity was also detected in extracts. Subcellular fractionation indicated that the glucosyltransferase and esterase activities are predominantly if not solely cytosolic. Taken together, these results show that reversible formation of pABA-Glc in the cytosol is interposed between pABA production in chloroplasts and pABA consumption in mitochondria. As pABA is a hydrophobic weak acid, its uncharged form is membrane-permeant, and its anion is consequently prone to distribute itself spontaneously among subcellular compartments according to their pH. Esterification of pABA may eliminate such errant behavior and provide a readily reclaimable storage form of pABA as well as a substrate for membrane transporters.Tetrahydrofolate and its derivatives, collectively termed folates, are essential cofactors for one-carbon reactions in eukaryotes and most prokaryotes. Bacteria, fungi, and plants synthesize folates de novo, but humans and other higher animals do not and therefore need a dietary supply (1, 2). For humans, a major part of this supply comes from plants, and there is consequently interest in engineering food plants for enhanced folate content (3, 4). This engineering approach to human nutrition, termed biofortification, depends on understanding the synthesis, metabolism, and transport of folates and their precursors in plants.The folate molecule is tripartite, comprising pteridine, glutamate, and p-aminobenzoate (pABA) 1 moieties (Fig. 1A). In plants, biochemical and genomic data indicate that the pteridine moiety is made in the cytosol, that the pABA moiety is made in the chloroplast, and that the two are coupled together and glutamylated in the mitochondrion (Fig. 1B) (reviewed in Refs. 5 and 6). In the course of folate synthesis, pABA must therefore exit chloroplasts, cross the cytosol, and enter mitochondria. This elaborate trajectory is not seen in bacteria and fungi, in which the entire pathway is cytosolic (7,8).Recent reviews of plant folate synthesis have tacitly assumed that pABA exists as the free acid in plant cells and that it moves between organelles in this form (5, 6). This assum...